Ethanol dependence of alpha1 antitrypsin c-terminal lys truncation by basic carboxypeptidases

ABSTRACT

The present invention provides methods of preparing alpha-1-antiproteinase inhibitor and controlling the amount of des-lys alpha-1-antiproteinase inhibitor in the preparation, and compositions comprising the same, as well as methods of treatment using the same.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Ser. No. 60/801,644,filed May 19, 2006, herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable.

BACKGROUND OF THE INVENTION

The invention relates to compositions of alpha1-proteinase inhibitor(A1PI) and methods of making and use.

Mature alpha1-proteinase inhibitor (A1PI) is a single chain glycoproteincomposed of 349 amino acids and 12% carbohydrate (weight %) (see, e.g.,Coan et al., Vox Sanguinis, 48:333-342 (1985)). Heterogeneity of A1PI isdue to posttranslational modifications and covalent linkage of 3 complexN-glycans to asparagines 46, 83 and 247^(1,2). The high negative chargeof A1PI is the result of multiple sialic acid residues on N-glycansleading to multiple isoforms of A1PI (M1 to M8) that become visibleafter electrophoresis³⁻⁸. Two minor cathodal isoforms, M7 and M8, arethe result of N-terminal truncation of 5 amino acids includingnegatively charged glutamic and aspartic acids⁹.

A1PI belongs to the family of serpins that inhibit serine proteases.Neutrophil elastase, an enzyme which degrades a number of proteins ofthe interstitial extracellular matrix, is a serine protease that isinhibited by A1PI. In patients with inherited A1PI deficiency thebalance between neutrophil elastase and A1PI is disturbed, whichincreases their risk of developing lung emphysema. In these patients,elastase released from neutrophils in the lower respiratory tractescapes neutralization by A1PI with consequent chronic destruction oflung parenchyma, which becomes clinically apparent in the third tofourth decade of life¹⁰.

To slow down the progression of emphysema in patients with A1PIdeficiency the protease—anti-protease balance is restored by life-longaugmentation therapy with highly purified plasma-derived A1PIconcentrates which raise A1PI in the circulation¹¹. Three differentproducts (Prolastin, Aralast, and Zemaira) are approved by the US FDAfor the treatment of A1PI deficiency. These products are manufacturedfrom large pools of ˜10000 liters of human plasma¹²⁻¹⁴. Upstreammanufacturing and downstream purification processes includingpathogen-reduction steps vary to differing extents betweenproducts^(12,15,16). After removal of the immune-globulin containingplasma fractions various sequential steps of the Cohn/ethanolfractionation, including chromatography, protein precipitation andco-precipitation followed by resolubilization, diafiltration for bufferexchange, concentration steps and viral reduction steps, take advantageof the physicochemical properties of A1PI to concentrate A1PI into anintermediate fraction. This fraction is used for subsequent downstreampurification. A1PI is therefore exposed to different physicochemicalconditions and to a variety of enzymes during the manufacturing process.

Available A1PI concentrates have a purity of >80% and specificactivities ranging from 0.6 to 1.0 U A1PI/mg protein with differentplasma protein impurity profiles. High resolution isoelectric focusing(IEF) analysis of A1PI present in A1PI products has revealed differencesin the IEF band pattern of glycolsoforms and raised questions frompatients, physicians and the FDA(www.fda.gov/ohrms/dockets/ac/05/transcripts/2005-4190t2.htm). Thisdifference in electrophoretic mobility was not caused by differences inN-glycan profiles, but mainly by varying degrees of C-terminal lysinetruncation at position 394 from the A1PI molecule adding an additionalnegative charge to the protein^(8,17,18),(http://www.fda.gov/ohrms/dockets/ac/05/transcripts/2005-4190t2.htm).The percent of A1PI C-terminal truncation as compared to total A1PIprotein differed in the three approved products. Aralast showedapproximately 60% (67%) truncated A1PI, while Prolast showed 2%truncated A1PI and Zemaira showed 6% truncated A1PI.

Basic carboxypeptidases are a group of enzymes that specifically cleaveC-terminal basic amino acids (arginine or lysine) from peptides andproteins leading to an increased negative charge of the protein¹⁹. Basiccarboxypeptidases are involved in a variety of biological processes suchas food digestion, inactivation of complement components²⁰, inhibitionof fibrinolysis²¹ and processing of peptide hormones²².

In this application, we show that A1PI belongs to the group of proteinsthat are a substrate for basic carboxypeptidases and that the C-terminaltruncated form of A1PI also occurs naturally. We also describe themanufacturing conditions which result in the removal of the positivelycharged C-terminal lysine of A1PI by carboxypeptidases.

SUMMARY OF THE INVENTION

Thus, it is an object of the present invention to provide A1PIcompositions with altered amounts of C-terminal lysine cleavage, methodsof making the same, and methods of treatment using the same.

The present invention provides, in one aspect, a method of controllingthe amount of des-lys alpha-1-proteinase inhibitor in analpha-1-proteinase inhibitor composition derived from human plasma, themethod comprising the step of altering the concentration of ethanol forCohn fractions IV-1+IV-4. In one embodiment, the amount of des-lysalpha-1-proteinase inhibitor is lowered or raised.

In another embodiment, the amount of des-lys alpha-1-proteinaseinhibitor is less that about 65% but more than about 2% of totalalpha-1-proteinase inhibitor in the composition. In another embodiment,the amount of des-lys alpha-1-proteinase inhibitor is less than about65% but more than about 6%. In another embodiment, the amount of des-lysalpha-1-proteinase inhibitor is less than about 6% but more than about2%.

In another embodiment, the amount of des-lys alpha-1-proteinaseinhibitor is more than about 70% of total alpha-1-proteinase inhibitorin the composition. In another embodiment, the amount of des-lysalpha-1-proteinase inhibitor is more than about 75% of totalalpha-1-proteinase inhibitor in the composition.

In another embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is about 50%. In another embodiment, theconcentration of ethanol used to precipitate Cohn fractions IV orIV-1+IV-4 is about 40%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than50% but more than 10%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than50% but more than 30%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than45% but more than 35%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than40% but more than 15%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than40% but more than 20%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than40% but more than 25%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than40% but more than 30%. In another embodiment, the concentration ofethanol used to precipitate Cohn fractions IV or IV-1+IV-4 is less than40% but more than 35%.

In another embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is less than 40% but more than 10%. Inanother embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is less than 35% but more than 10%. Inanother embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is less than 30% but more than 10%. Inanother embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is less than 25% but more than 10%. Inanother embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is less than 20% but more than 10%. Inanother embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is less than 15% but more than 10%. Inanother embodiment, the concentration of ethanol used to precipitateCohn fractions IV or IV-1+IV-4 is about 10%. In another embodiment, thepH of Cohn fractions IV-1 or IV-1+IV-4 is less than about pH 5.9. Inanother embodiment, the pH of Cohn fractions IV-1 or IV-1+IV-4 is morethan about pH 5.9.

In another aspect, the present invention provides a method of increasingthe amount of des-lys alpha-1-proteinase inhibitor in analpha-1-proteinase inhibitor composition derived from human plasma, themethod comprising the step of modulating the ethanol content of aprecipitate comprising alpha-1-proteinase inhibitor, wherein theprecipitate is selected from the group consisting of the Cohn IV-1precipitate or the Cohn IV-1+IV-4 precipitate. In one embodiment, theethanol content of the precipitate is less than 50% but greater than10%. In another embodiment, the ethanol content of the precipitate isless than 50% but greater than 30%. In another embodiment, the ethanolcontent of the precipitate is less than 45% but greater than 35%. Inanother embodiment, the ethanol content of the precipitate is about 40%.In another embodiment, an amount of carboxypeptidase suitable to cleavethe C-terminal lysine of alpha-1-proteinase inhibitor is added to theprecipitate. In another embodiment, the carboxypeptidase is selectedfrom the group consisting of carboxypeptidase N, carboxypeptidase U,carboxypeptidase M, or carboxypeptidase B.

In another aspect, the present invention provides a method of decreasingthe amount of des-lys alpha-1-proteinase inhibitor in analpha-1-proteinase inhibitor composition derived from human plasma, themethod comprising the step of modulating the ethanol content of aprecipitate comprising alpha-1-proteinase inhibitor, wherein theprecipitate is selected from the group consisting of the Cohn IV-1precipitate or the Cohn IV-1+IV-4 precipitate, and wherein the ethanolcontent of the precipitate is below 10%.

In another aspect, the present invention provides a method of increasingthe amount of des-lys alpha-1-proteinase inhibitor in analpha-1-proteinase inhibitor composition, the method comprising the stepof adding to the composition an amount of carboxypeptidase suitable tocleave the C-terminal lysine of alpha-1-proteinase inhibitor. In oneembodiment, the composition is derived from human plasma and is aprecipitate selected from the group consisting of the Cohn IV-1precipitate or the Cohn IV-1+IV-4 precipitate. In another embodiment,the carboxypeptidase is selected from the group consisting ofcarboxypeptidase N, carboxypeptidase U, carboxypeptidase M, orcarboxypeptidase B. In another embodiment, the method further comprisesthe step of modulating the ethanol content of the composition, whereinthe ethanol content is more than 10%. In another embodiment, the ethanolcontent is more than 10% but less than 50%. In another embodiment, theethanol content is more than 10% but less than 40%. In anotherembodiment, the ethanol content is more than 30% but less than 50%. Inanother embodiment, the ethanol content is more than 35% but less than45%. In another embodiment, the ethanol content is 40%.

In another aspect, the invention provides an alpha-1-proteinaseinhibitor composition comprising a physiologically acceptable carrierand an amount of des-lys alpha-1-proteinase inhibitor that is less thanabout 65% but more than about 2% of total alpha-1-proteinase inhibitorin the composition. In another embodiment, the amount of des-lysalpha-1-proteinase inhibitor is less than about 65% but more than about6%. In another embodiment, the amount of des-lys alpha-1-proteinaseinhibitor is less than about 6% but more than about 2%. In anotheraspect, the invention provides an alpha-1-proteinase inhibitorcomposition comprising a physiologically acceptable carrier and anamount of des-lys alpha-1-proteinase inhibitor that is more than about70-75% of total alpha-1-proteinase inhibitor in the composition.

In another aspect, the invention provides a method of treating familialemphysema, the method comprising administering a therapeuticallyeffective amount of a composition comprising a physiologicallyacceptable carrier and an amount of des-lys alpha-1-proteinase inhibitorthat is less than about 65% but more than about 2% of totalalpha-1-proteinase inhibitor in the composition. In another embodiment,the amount of des-lys alpha-1-proteinase inhibitor is less than about65% but more than about 6%. In another embodiment, the amount of des-lysalpha-1-proteinase inhibitor is less than about 6% but more than about2%. In another aspect, the invention provides a method of treatingfamilial emphysema, the method comprising administering atherapeutically effective amount of a composition comprising aphysiologically acceptable carrier and an amount of des-lysalpha-1-proteinase inhibitor that is more than about 70-75% of totalalpha-1-proteinase inhibitor in the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be explained in more detail by way of thefollowing Examples and drawing figures, to which, however, it shall notbe restricted.

FIG. 1: Effect of different CPs on the IEF pattern of A1PI: PPL: normalplasma pool, P: Prolastin, A: Aralast; C: non-truncated A1PI. 1, CPB(1.3 U/ml), 2, CPN (2.7 U/ml), 3, CPU (1 U/ml), 4, rCPM (0.25 U/ml).Anode is on the top. The non-truncated A1PI preparation was incubated at10 μM with different CPs without and with ethanol as indicated. Sampleswere loaded at 10 μM A1PI. The N-glycan structures of M6 and M4 and ofthe C-terminal truncated M6 and M4 in Aralast are symbolized by bi- andtri-antennary structures.

FIG. 2: A1PI cleavage at varying CPN and rCPM concentrations. A:Aralast, and C: non-truncated A1PI controls that were not treated withcarboxypeptidase. The non-truncated A1PI (10 μM) was incubated aftertemporary exposure to 40% EtOH with (A) CPN (1-270 mU/ml) or (B) rCPM(1-250 mU/ml). Anode is on the top. The bar chart shows the results ofthe densitometric evaluation of the gel. The relative amount ofnon-cleaved bands M6 and M4 is shown as a percent of the sum ofnon-cleaved and C-terminal cleaved M6 and M4, respectively. Arrows inFIG. 2 b indicate the pI shift caused by the C-terminal truncationaffecting also the minor bands M8 and M7.

FIG. 3: Effect of CP inhibitors on C-terminal lysine truncation ofdissolved IV-1 paste that had been in temporary contact with 40%ethanol. P, Prolastin, A, Aralast. Anode is on the top. Ethanol treatedIV-1 paste was dissolved in buffer (pH 8.8) and incubated during 6 h at22° C. in the presence of two distinct CP inhibitors. 1: in the presenceof 10% EtOH; 2: 1 h in the presence 40% EtOH, then dilution to 10%; 2a:+100 mM 6-Amino caproic acid; 2b, 2c, 2d: +10 μM, +100 nM, +1 nM of amore specific CPN inhibitor2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid, respectively.

FIG. 4: Effect of ethanol on the CPN-catalysed removal of C-terminallysine from A1PI. Prolastin or Aralast were mixed with different ethanolconcentrations and incubated with CPN during 10 min. Released lysine wasquantified by RP-HPLC relative to an internal standard.

FIG. 5: Effect of ethanol on the activity of CPN. The activity ofpurified CPN (diamonds) or plasma (full squares) was measured atincreasing ethanol concentrations and given as a percent of the initialactivity. Activity was measured using Hip-Arg as substrate and thereleased hippuric acid was quantified by RP-HPLC relative to an o-methylhippuric acid standard.

FIG. 6: Detection of C-terminal truncated A1PI in a human BAL solution.(a) Immunoblots after IEF of BAL solutions: P, Prolastin, A, Aralast; 1,2 and 3, human BAL samples. The IEF pattern of the corresponding plasmaof BAL 3 is also shown. (B) MS spectrum with C-terminal peptides ofA1PI. The peptide with m/z 657.40 representing the C-terminal truncatedpeptide VVNPTQ is highlighted.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Patients with hereditary emphysema are treated with alpha1-proteinaseinhibitor (A1PI) concentrates (also known as alpha1-antitrypsin). Adeficiency in A1PI represents one of the most common lethal hereditarydisorders of Caucasians in the United States and Europe. High resolutionisoelectric focusing (IEF) analysis of A1PI show commercial products andplasma have different glycolsoform band patterns. The banding patternsreflect an anodic shift of glycolsoforms resulting fromcarboxypeptidases cleaving off the positively charged C-terminal lysineresidue of A1PI. We showed that contact with ethanol during manufacturerenders A1PI susceptible to the cleaving with the extent of Lystruncation depending on the ethanol concentration. Furthermore incontrast to cell-free systems, A1PI in broncho-alveolar lavage fluid isalso partly Lys truncated. This is probably due to the presence oflipid-anchored carboxypeptidase M in lung tissue. Lys truncation in A1PIis therefore not only associated with manufacturing processes but isalso a physiologic process.

Thus, modulation of the amount of the des-Lys form of A1PI is possible.Without being limited by theory, in one embodiment, lower amounts of thedes-Lys form are desirable, for example, to improve serum stability andhalf life. In one embodiment, higher amounts of the des-Lys form aredesirable, for example, in the case where the A1PI is administered byinhaling. A1PI in serum typically contains the C-terminal lysine, whilethe form present in the lung has a greater proportion of the des-Lysform.

II. Definitions

“Cohn fractionation” refers to the Cohn-Oncley fractionation procedurefor human plasma. See, e.g., E. J. Cohn, et al., J. Amer. Chem. Soc.,68, 459 (1946); E. J. Cohn, U.S. Pat. No. 2,390,074; and Oncley, et al.,J. Amer. Chem. Soc., 71, 541 (1949) the entire disclosures of which arehereby incorporated by reference herein. See also U.S. Pat. No.6,284,874. The Cohn-Oncley process involves a series of cold ethanolprecipitation steps during which specific proteins are separatedaccording to isoelectric point by adjusting pH, ionic strength, proteinconcentration, temperature and ethanol concentration. See also U.S. Pat.No. 6,284,874

By “therapeutically effective amount or dose” or “sufficient amount ordose” herein is meant a dose that produces effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). The dose can beadministered parenterally, e.g., intravenously, or by inhalation.

“Hereditary emphysema” or “familial emphysema” refers to a genetic lungdisease caused by A1PI deficiency. A1PI deficiency is also related toasthma, chronic obstructive pulmonary disease (COPD), chronicbronchitis, and broncheictasis. Patients with familial emphysema may bediagnosed or misdiagnosed with these disorders. Treatment of thesedisorders with A1PI is also contemplated by the present invention.

III. Methods of Making A1PI

A1PI is purified from an impure protein fraction. The impure proteinfraction may be plasma, A1PI produced by recombinant methods or anyother source comprising A1PI protein. In one embodiment A1PI is preparedfrom frozen plasma. The plasma is thawed and the Cohn IV-1 precipitateor the Cohn IV-1+IV-4 precipitate is prepared. The preparation of theCohn IV-1 and the Cohn IV-1+IV-4 fraction are well known in the art andis described in U.S. Pat. No. 6,284,874 (herein incorporated byreference), with modifications as described herein to modulate theamount of des-lys A1PI in the final preparation. Other methods of makingA1PI are described, e.g., in U.S. Pat. No. 6,974,792. A1PI has also beenproduced recombinantly (see, e.g., Courtney, M. et al, High-LevelProduction of Biologically Active Human Alpha-1-Antitrypsin inEscherichia coli, Proc. Natl. Acad. Sci. USA 81: 669-673 (1984)); Sleep,D. et al., Saccharomyces cerevisiae Strains that Over ExpressHeterologous Proteins, Bio/Technol. 9: 183-187 (1991); Bischoff et al.,Purification & Biochemical Characterization of Recombinant Alpha 1lAntitrypsin Variant Expressed in Escherichia coli, Biochemistry30:3464-3472 (1991)).

In particular embodiments of the invention, the level of des-lys A1PI inthe final preparation is controlled by modulating the ethanol content ofthe Cohn IV-1 precipitate or the Cohn IV-1+IV-4 precipitate. In someembodiments of the invention in which the level of des-lys A1PI isincreased, the ethanol content is increased by treating the precipitatewith ethanol in a concentration between 10% and 50%, or between 30% and50%, or between 35% and 45%, or about 40%. In some other embodiments ofthe invention in which the level of des-lys A1PI is decreased, theethanol content is maintained below 10%. In other embodiments of theinvention in which the level of des-lys A1PI is increased, an A1PIcontaining composition, such as the precipitate, is treated under anappropriate ethanol concentration with a basic carboxypeptidase, such ascarboxypeptidase N(CPN²³; EC 3.4.17.3), carboxypeptidase U (CPU²⁴; EC3.4.17.20), carboxypeptidase M (CPM²⁵; EC 3.4.17.12) andcarboxypeptidase B (CPB²⁶; EC 3.4.17.2)

IV. A1PI Activity Assays

In one embodiment, a chromogenic assay is used to detect A1PI activity.The assay utilizes a trypsin sensitive chromogenic substrate whichreleases p-nitroaniline in the presence of trypsin (supplied by SigmaChemical Co. of St Louis, Mo.). The p-nitroaniline released is detectedat 405 nm. A1PI inhibits the release of p-nitroaniline from thesubstrate. The activity of A1PI in the product can be determined byreference to a standard A1PI activity curve. Other assays are known inthe art and can be used to evaluate activity.

V. Protein Content

Protein content is determined by a BIO-RAD assay method utilizingdifferential color change of a Coomassie Blue dye in response to variousconcentrations of protein measured at 595 nm. The protein content iscalculated from a standard curve. Other assays are known in the art andcan be used to determine protein content.

VI. Administration

A1PI is infused into a patient at a rate of about 0.08 ml/kg body weightper minute for the first 10 minutes. If the patient does not experienceany discomfort, the rate is increased as tolerated. If tolerated,subsequent infusions to the same patient may be at the higher rate. Ifadverse events occur, the rate should be reduced or the infusioninterrupted until the symptoms subside. The infusion may then be resumedat a rate which is tolerated by the patient. If large doses are to beadministered, several reconstituted vials of A1PI may be pooled in anempty, sterile I.V. infusion container using aseptic technique.

In another embodiment, A1PI can be administered nasally and/or orally,by inhaling from a nebulizer or similar apparatus.

VII. EXAMPLES Example 1

A. Results

IEF Isoform Band Pattern of Human A1PI

The IEF isoform band pattern of human plasmatic A1PI is determinedmostly by the neuraminic acid content of their 3 N-linkedcarbohydrates⁵⁻⁸ and by the extent of C-terminal Lys truncation^(17,18).M4 and M6 are the predominant bands in plasma and in the licensed A1PIproduct Prolastin. They contain two biantennary and one triantennarysugar side chain(s) (M4) or three biantennary sugar structures (M6)(FIG. 1). In the case of Aralast, minor amounts of the M4 and M6 bandswere still visible and the major bands, which were slightly less acidicthan the original M2 and M4 bands, were visible in the M2 and M4 region.As the ratio of different complex sugar structures in all products werealmost identical, a C-terminal Lys truncation was identified as thecause for this anodal band shift^(17,18).

Purified A1PI

We found that basic carboxypeptidases such as carboxypeptidase N(CPN²³;EC 3.4.17.3), carboxypeptidase U (CPU²⁴; EC 3.4.17.20), carboxypeptidaseM (CPM²⁵; EC 3.4.17.12) and carboxypeptidase B (CPB²⁶; EC 3.4.17.2)could indeed cleave off C-terminal lysine from a non-cleaved A1PIproduct. C-terminal lys removal was particularly observed when the A1PIpreparation had been in temporary contact with 40% ethanol followed bydilution to 10% ethanol (CPN, CPU, CPM, CPB) or when A1PI was incubatedwith the carboxypeptidase in the presence of only 10% ethanol (CPB andslightly for CPN) (FIG. 1). Carboxypeptidase treatment in the absence ofethanol did not induce a shift of the major IEF bands M6 and M4 to theM4 and M2 region, respectively. Whereas CPB, CPU and CPM caused almostcomplete Lys truncation of A1PI that had been in temporary contact with40% ethanol, only ˜60% truncation was observed with CPN, a level whichis characteristic for Aralast.

The concentration dependency of C-terminal Lys truncation of 40%ethanol-pretreated purified A1PI was determined for CPN and CPM. Wefound that 50 mU CPM/ml or between 50 and 270 mU CPN/ml could induce 50%C-terminal A1PI truncation (FIG. 2A+B). For comparison, about 65 mUCPN/ml 27 is detected in plasma corresponding to a plasma concentrationof 30 μg/mL, 40 mU/ml in the starting material of the Cohn fractionationprocess and a similar amount of CPN is present in the starting materialof Aralast. An anodic shift of the A1PI isoforms M7 and M8 was alsoinduced by CPM (FIG. 2B).

A1PI in Ethanol Precipitates or Pastes

Purified A1PI preparations and A1PI present in dissolved alcoholprecipitates, which are the starting material for all plasmatic A1PIdownstream processes, appeared to have a similar ethanol dependence ofLys truncation by carboxypeptidases.

So-called Cohn IV-1 precipitate²⁸ was the starting material for A1PIproducts with a plasma-like IEF pattern. Cohn IV-1 precipitate isusually derived after ethanol fractionation of plasma by precipitationwith 20% ethanol at pH 5.2.

A somewhat different alcohol precipitation was used for Aralast. Afterinitial precipitation of plasma with 20% ethanol at pH 5.2, theprecipitate (the so-called IV-1 paste) was not removed and the 20%ethanol-containing plasma suspension was adjusted to pH 5.9 and then to40% ethanol, which resulted in a second precipitation (the so-calledIV-4 paste). The total precipitate, the (IV₁₊₄) paste, was collected andserved as the starting material for the Aralast process.

Based on our experiences with purified A1PI, prior exposure of IV₁₊₄paste to 40% ethanol might have caused the truncation in the subsequentextraction step. This hypothesis was therefore tested with IV-1 pastewhich contained almost identical levels of CPN (59 mU/ml) compared toIV₁₊₄ paste (61 mU/ml, both normalized to direct paste extract with 7volumes).

IV-1 paste was dissolved in buffer at pH 8.8 and was temporarily exposedto 40% ethanol, diluted to 10% ethanol and then stored during 6 hours(40=>10% EtOH). This procedure was sufficient for C-terminal Lystruncation at a level similar to Aralast and was apparently induced bythe CPN present in the paste (FIG. 3). Temporary contact of dissolvedIV-1 paste with 10% ethanol did not result in Lys truncation. The Lystruncation induced by the procedure used (40=>10% EtOH) could beinhibited by 6-amino caproic acid²⁹ and dose-dependently byDL-2-mercaptomethyl-3-guanidinoethylthio-propanoic acid, a more specificCPN-inhibitor³⁰ (CPU Ki=0.20 μM; CPN Ki=0.0087 μM³¹).

As observed for purified A1PI (FIG. 1) CPB added to dissolved IV-1 pastecould also induce almost complete Lys truncation in the presence of only10% ethanol, whereas the CPN present in the paste (in the absence ofCPB) did not remove the C-terminal lysine in the presence of 10% ethanol(data not shown).

Ethanol Dependency of Lys Truncation in Purified A1PI

Lys truncation in purified A1PI was also measured by quantification ofreleased Lys using a HPLC method. This is a sensitive method that wasused to determine the alcohol dependence of Lys truncation of Prolastinor Aralast by CPN. Both A1PI products showed a linear increase of Lysremoval with increasing ethanol concentration starting at 5% ethanol forProlastin and at 10% for Aralast (FIG. 4). Differences in the ethanoldependency between the products apparently reflects their differentstarting level of truncation. This method also showed that very smallamounts of Lys were removed from Prolastin in the absence of ethanol.

Apart from the possible influence of ethanol on A1PI, a direct effect ofethanol on CPN, i.e. on the rate of the substrate (Bz-Gly-Arg) cleavagecould be detected, which almost doubled at 10% ethanol (FIG. 5). Thiseffect was reversible and completely disappeared after dilution.

BAL Samples

Bronchio-alveolar lavage (BAL) fluid was isolated from patients who hadnot received A1PI augmentation therapy. BAL samples, which werecharacterized by high resolution IEF, showed the IEF patterncharacteristic for C-terminal lysine removal but at a lower level thanthat in Aralast (FIG. 6). This suggests that CPM present in lung tissue32 might have been responsible for the lysine removal under physiologicconditions. A corresponding plasma sample did not show this partial Lystruncation.

The Lys-cleavage of A1PI in BAL samples was confirmed by MS analysis oftryptic peptides, which demonstrated that the C-terminal lysine wasremoved from the A1PI C-terminal peptide at 657 m/z.

B. Discussion

Contributors to the heterogeneity of a biological product can includebiosynthetic mechanisms used by living organisms, manufacturingprocesses, and storage conditions. Heterogeneity in plasma-derivedtherapeutic proteins is only slightly affected by contributions fromindividual donor variations because of the dilution effect provided bythe large plasma pool size of several thousand donors.

We have shown that large-scale manufacturing conditions of A1PI causeprimary sequence modifications of A1PI resulting in structuralheterogeneity. Fractionation with cold ethanol²⁸ used in the large-scalemanufacturing of A1PI facilitates removal of the C-terminal lysine fromthe A1PI molecule by carboxypeptidases. The loss of approximately 60% ofthe total C-terminal lysine content of A1PI in Aralast gives themolecule an extra negative charge causing an unusual mobility of theglycolsoforms with an anodal shift on IEF gels. Differences in theextent of C-terminal lysine truncation between the commercial productsis due to variations in the manufacturing conditions exposing A1PI todifferent ethanol concentrations, and not to differences in theconcentration of carboxypeptidases present in the various intermediatefractions. Prolastin, which has a C-terminal truncation of about 2%, ispurified from fraction IV-1: this fraction is obtained by precipitationof fraction I supernatant with 20% ethanol. Aralast is obtained fromfraction IV-1+IV-4, which is made by increasing the ethanolconcentration of fraction IV-1 to 40%, without preceding removal of theprecipitate. Prior exposure to high concentrations of ethanol isrequired to make A1PI more susceptible to carboxypeptidases.

The data presented support the hypothesis that A1PI has first to bealtered by temporary exposure to 40% ethanol before the C-terminallysine becomes susceptible to the action of basic carboxypeptidases to amajor extent. In contrast, many other proteins can be C-terminaltruncated under physiological conditions without prior alteration(probably conformational change) of the protein (immunoglobulin^(33,34),alpha2 antiplasmin³⁵, erythropoietin³⁶, hemoglobin³⁷, creatine kinase³⁸,enolase³⁹, complement factor⁴⁰, albumin⁴¹, tissue plasminogenactivator¹⁹, stromal cell-derived factor 1 alpha⁴²). The alteration ofA1PI induced by temporary exposure to 40% ethanol persists even afterdilution to 10% ethanol (FIG. 3). Exposure of A1PI to only 10% ethanolis not sufficient to release a major amount of C-terminal lysine by theaction of CPN required to produce a visible anodal shift ofglycolsoforms. Only the small 35 kDa carboxypeptidase B from pancreas,which is not found in plasma, was able to completely truncate A1PI inthe presence of 10% ethanol.

The ethanol-induced structural alteration of A1PI may also beresponsible for the different behavior of alcohol precipitates IV-1 andIV-1+4 as starting material of an otherwise almost identical A1PIdownstream process. IV-1+4 paste, which has been in contact with 40%ethanol, yields a final product with ˜60% lysine truncation, whereasIV-1 paste, generated with 20% ethanol, after using identical downstreamprocessing steps, yields a final A1PI product with only 2% lysinetruncation (qualitatively shown in FIG. 1, Control; compare A with C).

In a purified system, CPN in a dose-dependent fashion changed theglycolsoform pattern of A1PI made from fraction IV-1 into a band patternidentical to that of A1PI made from IV-1+4. The pronounced anodal shiftof the original M6 and M4 bands to the M4 and M2 region is mainly causedby temporary exposure of A1PI to 40% ethanol.

In plasma, only 2 basic carboxypeptidases are known: CPN, which isconstitutively active, and proCPU, which requires proteolyticactivation. CPN is probably the carboxypeptidase responsible for theA1PI C-terminal truncation during extraction of the various pastes. CPNis found in similar amounts in all alcohol precipitates studied and thelysine removal from A1PI can be completely inhibited by addition of 100mM 6-aminocaproic acid or by the more CPN-specific inhibitor2-mercaptomethyl-3-guanidinoethylthiopropanoic acid³⁰ at 10 μmol/l⁴³.

However, CPU is not stable during incubation at 37° C. for 1 h, whereasthe carboxypeptidase that is responsible for A1PI truncation withstandssuch treatment.

This finding of almost complete inhibition of truncation demonstratesthat no truncation has taken place during ethanol fractionation, aslysine removal requires both prior exposure of A1PI to ethanol as wellas a pH>5.9 to provide optimal conditions for CPs activity.

CPN is a zinc-dependent metalloproteinase that is synthesized in theliver and secreted into the blood. It is a glycoprotein that consists oftwo 83-kDA regulatory subunits and two 50-kDA catalytic subunits whichform a stable tetramer of 280-kDa⁴⁴. CPN was originally identified to beresponsible for inactivating bradykinin by removal of C-terminalarginine. CPN also removes C-terminal arginines from the anaphylatoxinsC3a, C4a, and C5a with subsequent reduction of their biologicalactivity⁴⁰. Creatine kinase MM, an intracellular enzyme that mediatesthe transfer of a phosphate group from adenosine triphosphate tocreatinine, is another substrate for CPN. CPN cleaves C-terminal lysinesfrom CK-MM to generate CK-MM1 and CK-MM2⁴⁵. The CPN activity in plasmais about 65 mU/ml which is sufficient for lysine removal from severalimportant physiological proteins like erythropoietin³⁶, hemoglobin⁴⁶,stromal cell-derived factor-1-alpha⁴⁷, complement proteins⁴⁰, albumin⁴¹and probably many other proteins with a basic C-terminus. 50 mU CPN/mlcleave a 10-20% of the C-terminal lysines from purified uncleaved A1PI(FIG. 2).

On the other hand, using a direct measurement of lysine cleavage, wedemonstrated that CPN is able to remove lysine to a small extent even inthe absence of ethanol, and that this reaction can be enhanced byethanol. However, the small amount of des-Lys A1PI formed in the absenceof ethanol probably escapes visualization on IEF gels due to limits inthe resolution. Apart from having an influence on the substrate, ethanolseems also to directly affect carboxypeptidase activity. Increasing theethanol concentration, linearly increases CPN activity (measured withHip-Arg) resulting in a doubled CPN activity at a final ethanolconcentration of 10%. An effect of ethanol on CPB activity toward smallsynthetic substrates also has been described⁴⁸.

Although with the current methods we were unable to show that thedes-Lys form of A1PI is present in plasma, A1PI recovered from human BALsamples consists in part of the des-Lys form. This is most likely due tothe action of CPM which is highly expressed in lung tissue³², and CPMactivity is also found in bronchio-alveolar fluid⁴⁹. We speculate thatA1PI is exposed to CPM when it diffuses through lung tissue from thevasculature into the alveolar space. CPM, attached to lung membranes bya phosphatidylinositol anchor is also found in alveolar type 1 cells,which comprise about 9% of total human lung cells and 93% of totalalveolar epithelial surface area³². Type I cells, together with basementmembrane and capillary endothelial cells, function primarily as a thin,gas-permeable membrane between air and blood.

In summary, we have identified the cause of C-terminal truncation ofA1PI present in products used for augmentation therapy and shown thatA1PI becomes a substrate for carboxypeptidase, specifically CPN, due tothe exposure of A1PI to ethanol during Cohn fractionation. In search fora naturally occurring des-Lys form of A1PI we discovered the presence ofthis modification in BAL from patients who had not been treated with anyof the products.

Methods

Reagents

Carboxypeptidase B (Sigma), carboxypeptidase N (Elastin Company),carboxypeptidase U (proCPU, purified and activated according to theprotocol described⁵⁰) and a recombinant carboxypeptidase M expressed inP. pastoris, and purified as described⁵¹ were used. The commerciallyavailable A1PI concentrates, Aralast and Prolastin, lot numbers LH020A31and PR4HA43, respectively, as well as a purified A1PI preparation(prepared according to the ARALAST process but with IV-1 paste asstarting material) were used. The various reagents used will bedescribed below. Unless stated otherwise, reagents of the highest purityavailable were used.

Measurement of Basic Carboxypeptidases

The activity of the basic carboxypeptidases was measured with aHPLC-assisted assay as described⁵². This assay is based on the cleavageof the synthetic substrate hippuryl-L-arginine. Released hippuric acidwas determined with reverse phase high performance liquid chromatography(RP-HPLC). Briefly, to 10 μl of sample, 40 μl of 30 mMhippuryl-L-arginine (Bachem Feinchemikalien, Buchs, Switzerland) in 50mM HEPES, pH 8.0, was added and incubated for 30 minutes at 37° C. in awater bath. Hippuryl-L-arginine cleavage was stopped by adding 50 μl of1 M HCl. 10 μl of o-methyl hippuric acid (synthesized from methylbenzoylchloride) serving as an internal standard was added afterwards. Thehippuric acid (Bz-Gly, Fluka, Buchs, Switzerland) and o-methyl hippuricacid were extracted with ethyl acetate (300 μl). This layer wasevaporated to dryness, redissolved in the mobile phase (10 mM KH₂PO₄,10% acetonitrile, pH 3.5) and injected unto the column (C-18 Chromolith,performance 100-4.6 mm column (Merck, Darmstadt, Germany). Theseparation was done in isocratic mode and monitored at 228 nm. One unitof carboxypeptidase activity is defined as the amount of enzyme requiredto release 1 μmol of hippuric acid per min at 37° C. under the assayconditions described.

Incubation with CPs and Analysis by IEF

A1PI solutions were incubated with different CPs. Temporary exposure to40% ethanol was achieved by diluting the A1PI preparation (38 μM) with96% ethanol. The precipitate formed was kept 30 min at −20° C. and afurther 30 min at +4° C., before it was diluted with TRIS-HCl buffer (pH8.8) to a final concentration of 10% ethanol. Concomitantly, CPs wereadded with the TRIS buffer and the reaction mixture containing 10 μMA1PI was then incubated at 37° C. for 60 min. Before IEF analysisdithioerythritol (DTE) was added to a final concentration of 5 mM. Inaddition, the incubation with CPs was done without ethanol or at 10%ethanol. Furthermore, we ran two concentration rows with either CPN(270-1 mU/ml) or CPM (250-10 mU/ml) after temporary exposure of A1PI to40% ethanol (CPN units were tested against Hip-Arg).

HPLC Method for Determination of Liberated Lysine

The cleaved C-terminal lysine of A1PI was determined using automatedorthophtalicdialdehyd (OPA) pre-column derivatization andhigh-performance liquid chromatography. The derivatization reagent wasprepared by dissolving 100 mg of OPA in 2.5 mL of methanol by shortultrasonication, adding 23 mL of deoxygenated sodium borate buffer (0.2mol/L, pH 9.0), 100 μl of 2-mercaptoethanol and 100 μl of Brij 35 asdescribed⁵³. The system used was an ASTED HPLC system (Gilson, Paris)with Shimadzu RF-A fluorescence detector and a C-18 Chromolith ODS4.7*100 mm column. A mixture of 50 mM KH₂PO₄, acetonitrile and methanol(ratio 50:24:26) was used as mobile phase and ornithine was used asinternal standard.

To 30 μl 0.5 mM A1PI (using ARALAST® or PROLASTIN®) 10 μl water orethanol (different concentrations) was added. Afterwards 10 μl CPN (500U/L) was added and after a 10 minutes incubation interval the reactionwas stopped by adding 50 μl internal standard in 0.25 M HCl. The cleavedlysine was quantified as described above. The assay demonstrated goodlinearity between 2-120 μM final concentrations of Lys.

Effect of Ethanol on the Activity of Purified and Plasma CPN

The effect of increasing concentrations of ethanol on CPN activity wastested as follows. To 10 μl purified CPN or plasma 10 μl ethanol (0-40%,maximum final concentration therefore 20%) was added and directlyincubated with 30 μl substrate (30 mM Hip-Arg pH 8.0) for 20 minutes andactivity was quantified as described above.

Extraction of Pastes

40%=>10% EtOH:

Cohn IV-1 paste was dissolved in 2.6 volumes of 10 mM Tris-buffer at pH8.8 and incubated for 30 min at 22° C. The suspension was made 40% inethanol (by addition of 2.4 volumes of ethanol), stirred for a further60 min, diluted with the same Tris-buffer to 10% ethanol and extractedfor a further 6 h.

10% EtOH:

IV-1 paste was dissolved in 20.6 volumes of 10 mM Tris-buffer at pH 8.8and incubated during 30 min at 22° C. The suspension was made 10% inethanol (by addition of 2.4 volumes of ethanol) and stirred for afurther 6 h at 22° C. and pH 8.8.

The following inhibitors were added in parallel during extraction anddilution with Tris-buffer: 100 mM 6-aminocaproic acid, 10 μM or 1 nM2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (a more specificinhibitor of CPN). The samples were characterized by high resolutionisoelectric focusing.

Comparison of Different Pastes:

Pastes were dissolved in 24 volumes (potential presence of filter aidwas ignored) of 30 mM Tris/HCl buffer (pH 10.4), adjusted to pH 8.5 andstirred for 2 h at 4° C. and 1.5 h at 40° C. and cooled again to 20° C.Samples could be frozen and stored at −20° C.

Isoelectric Focusing

For high resolution isoelectric focusing a new hybrid IEF method usingIPG-Immobiline gels (GE Healthcare Bio-Sciences, pI 4.2-4.9, Uppsala,Sweden) was developed which is similar to that described by Weidinger⁵⁴.Samples were diluted with water to 0.5 mg A1PI/mL and 5 mM DTE wasadded. Samples (20 μl) were applied close to the cathode on thepre-focused gel and focused for 150 min (5000 Vh). For paste extractsfocusing time was extended to 420 min. Gels were stained with CoomassieBrilliant Blue G-250 (BioRad, Hercules, USA) as described by Neuhoff⁵⁵.Blotting and immunostaining were done with a rabbit anti-A1PI antibody(Rabbit Anti-Human Alpha-1-Antitrypsin, #A0012, DakoCytomation,Glostrup, Denmark).

BAL Samples

BAL samples from patients with chronic obstructive pulmonary disease(COPD) or cancer were provided by Prof. R. Sakalauskas, Vilnius,Lithuania. Informed consent was obtained from these individuals. Onlysamples with A1PI concentrations higher than 2 μg/mL were furtherinvestigated.

Isolation and MS Analysis of A1PI from BAL Fluid

To concentrate the proteins before SDS-PAGE, BAL samples were subjectedto a solid phase extraction (SPE) cartridge containing a C4 gel(Macherey-Nagel, Düren, Germany). The SPE cartridge was primed with 1 mLacteonitrile followed by 1 mL of water. All solvents used for priming,washing and elution of proteins contained 0.1% formic acid. The BALsample was loaded and the column was subsequently washed with 1 mL ofwater. Elution of proteins was achieved by applying 500 μl of 40%acetonitrile. The eluates were dried in a Speed Vac concentrator. Thedry samples were solved in 15 μl of SDS-PAGE loading buffer and thensubjected to SDS-PAGE under reducing conditions⁸. Coomassie-stainedbands were de-stained, carbamidomethylated, digested with trypsin andextracted from gel pieces. The extracts were dried in a Speed Vacconcentrator and reconstituted with water containing 0.1% formic acidbefore LC-MS analysis. MS and MSMS analysis was performed as describedpreviously. For every sample, both a MS and tandem MS run wereperformed.

Example 2

Isoelectric focusing (IEF) of alpha(1)-proteinase inhibitor (A1PI) showsthat commercial products and plasma have different glycolsoform bandpatterns. Those in Aralast reflect an anodal shift of glycolsoforms. Theprotein, including glycoproteomic analyses, and structural features ofA1PI products were investigated by state-of-the-art techniques.

Aralast, Prolastin, and Zemaira were analyzed by high-resolution IEF andhigh-performance size-exclusion chromatography (HP-SEC). Preparativeseparated isoforms from IEF were further purified by chromatography andsubjected to mass spectrometry for sequence analyses, peptide mapping,and glycosylation analysis. Deamidation was quantified by enzymaticisoaspartate detection. Multiple sequence alignments and structuralbioinformatics analyses were performed.

In HP-SEC, Prolastin had the highest aggregate content at approximately30 percent. Isoforms from all products purified by high-resolution IEFwere sequenced with an amino acid coverage of more than 98 percent.Deamidation of Asn116 and Asn314 in A1PI was to found to some extent inall products and confirmed quantitatively by enzymatic analysis. Therewere no signs of methionine oxidation. Cys232 was found to becysteinylated in A1PI in Prolastin and Aralast as in plasma, but not inZemaira. All products showed truncation of the C-terminal lysine. IntactA1PI concentrates contained mainly diantennary, disialylated and smalleramounts of triantennary, trisialylated N-glycans. The percentage offucosylation was similar in all products. Site-specific glycan analysisrevealed bands M6 contained only diantennary glycans, whereas the moreacidic bands M4 and M2 also carried triantennary structures. The mostacidic isoforms, M2 in Prolastin and Zemaira and M0 in Aralast,additionally exhibited tetraantennary N-glycans.

Protein chemical characterization of A1PI showed that all A1PI productsto some extent differ from A1PI circulating in human plasma.Bioinformatic analysis indicated that removal of C-terminal Lys394 andcysteinylation of Cys232 are unlikely to affect structure and/orfunction of A1PI but cysteinylation may influence interaction betweenA1PI and its physiologic ligands. Lack of cysteinylation may cause aprotein to dimerize. Aralast, Prolastin, and Zemaira contain the sameset of N-glycans in the same ratios as those in normal human plasmaA1PI. Tri- and tetraantennary structures are responsible for thepartitioning into IEF isoforms, with the migration shift of Aralast notbeing due to any difference in the N-glycosylation, but to the partialloss of the C-terminal lysine (des-Lys A1PI).

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of controlling or modulating the amount of des-lysalpha-1-proteinase inhibitor in an alpha-1-proteinase inhibitorcomposition derived from human plasma, the method comprising the step ofaltering the concentration of ethanol in the starting material fraction,wherein the starting material fraction is selected from the groupconsisting of Cohn fraction IV-1 and Cohn fraction IV-1+IV-4.
 2. Themethod of claim 1, wherein the amount of des-lys alpha-1-proteinaseinhibitor is lowered.
 3. The method of claim 2, wherein the amount ofdes-lys alpha-1-proteinase inhibitor in the composition is less thanabout 65% but more than about 2% of total alpha-1-proteinase inhibitorin the composition.
 4. The method of claim 2, wherein the amount ofdes-lys alpha-1-proteinase inhibitor in the composition is less thanabout 65% but more than about 6% of total alpha-1-proteinase inhibitorin the composition.
 5. The method of claim 2, wherein the amount ofdes-lys alpha-1-proteinase inhibitor in the composition is less thanabout 6% but more than about 2% of total alpha-1-proteinase inhibitor inthe composition.
 6. The method of claim 2, wherein the concentration ofethanol used to precipitate Cohn fractions IV-1+IV-4 is less than 40%but greater than 10%.
 7. The method of claim 2, wherein theconcentration of ethanol used to precipitate Cohn fractions IV-1+IV-4 isless than 35% but greater than 10%.
 8. The method of claim 2, whereinthe concentration of ethanol used to precipitate Cohn fractionsIV-1+IV-4 is less than 30% but greater than 10%.
 9. The method of claim2, wherein the concentration of ethanol used to precipitate Cohnfractions IV-1+IV-4 is less than 25% but greater than 10%.
 10. Themethod of claim 2, wherein the concentration of ethanol used toprecipitate Cohn fractions IV-1+IV-4 is less than 20% but greater than10%.
 11. The method of claim 2, wherein the concentration of ethanolused to precipitate Cohn fractions IV-1+IV-4 is less than 15% butgreater than 10%.
 12. The method of claim 2, wherein the pH of Cohnfractions IV-1+IV-4 is less than pH 5.9.
 13. The method of claim 1,wherein the amount of des-lys alpha-1-proteinase inhibitor is increased.14. The method of claim 13, wherein the amount of des-lysalpha-1-proteinase inhibitor in the composition is more than about 70%of total alpha-1-proteinase inhibitor in the composition.
 15. The methodof claim 13, wherein the amount of des-lys alpha-1-proteinase inhibitorin the composition is more than about 75% of total alpha-1-proteinaseinhibitor in the composition.
 16. The method of claim 13, wherein theconcentration of ethanol used to precipitate Cohn fractions IV-1+IV-4 isabout 50%.
 17. The method of claim 13, wherein the concentration ofethanol used to precipitate Cohn fractions IV-1+IV-4 is about 40%. 18.The method of claim 13, wherein the concentration of ethanol used toprecipitate Cohn fractions IV-1+IV-4 is less than 50% but greater than10%.
 19. The method of claim 13, wherein the concentration of ethanolused to precipitate Cohn fractions IV-1+IV-4 is less than 50% butgreater than 30%.
 20. The method of claim 13, wherein the concentrationof ethanol used to precipitate Cohn fractions IV-1+IV-4 is less than 45%but greater than 35%.
 21. The method of claim 13, wherein the pH of Cohnfractions IV-1+IV-4 is greater than about pH 5.9.
 22. A method ofincreasing the amount of des-lys alpha-1-proteinase inhibitor in analpha-1-proteinase inhibitor composition derived from human plasma, themethod comprising the step of modulating the ethanol content of aprecipitate comprising alpha-1-proteinase inhibitor, wherein theprecipitate is selected from the group consisting of the Cohn IV-1precipitate or the Cohn IV-1+IV-4 precipitate.
 23. The method of claim22, wherein the ethanol content of the precipitate is less than 50% butgreater than 10%.
 24. The method of claim 22, wherein the ethanolcontent of the precipitate is less than 50% but greater than 30%. 25.The method of claim 22, wherein the ethanol content of the precipitateis less than 45% but greater than 35%.
 26. The method of claim 22,wherein the ethanol content of the precipitate is about 40%.
 27. Themethod of claim 22, wherein an amount of carboxypeptidase suitable tocleave the C-terminal lysine of alpha-1-proteinase inhibitor is added tothe precipitate.
 28. The method of claim 27, wherein thecarboxypeptidase is selected from the group consisting ofcarboxypeptidase N, carboxypeptidase U, carboxypeptidase M, orcarboxypeptidase B.
 29. A method of increasing the amount of des-lysalpha-1-proteinase inhibitor in an alpha-1-proteinase inhibitorcomposition, the method comprising the step of adding to the compositionan amount of carboxypeptidase suitable to cleave the C-terminal lysineof alpha-1-proteinase inhibitor.
 30. The method of claim 29, wherein thecomposition is derived from human plasma and is a precipitate selectedfrom the group consisting of the Cohn IV-1 precipitate or the CohnIV-1+IV-4 precipitate.
 31. The method of claim 29, wherein thecarboxypeptidase is selected from the group consisting ofcarboxypeptidase N, carboxypeptidase U, carboxypeptidase M, orcarboxypeptidase B.
 32. The method of claim 29, further comprising thestep of modulating the ethanol content of the composition, wherein theethanol content is more than 10%.
 33. A method of decreasing the amountof des-lys alpha-1-proteinase inhibitor in an alpha-1-proteinaseinhibitor composition derived from human plasma, the method comprisingthe step of modulating the ethanol content of a precipitate comprisingalpha-1-proteinase inhibitor, wherein the precipitate is selected fromthe group consisting of the Cohn IV-1 precipitate or the Cohn IV-1+IV-4precipitate, and wherein the ethanol content of the precipitate is below10%.
 34. An alpha-1-proteinase inhibitor composition comprising aphysiologically acceptable carrier and an amount of des-lysalpha-1-proteinase inhibitor that is less than about 65% but more thanabout 2% of total alpha-1-proteinase inhibitor in the composition, thecomposition made using the method of claim
 1. 35. The composition ofclaim 34, wherein the amount of des-lys alpha-1-proteinase inhibitorthat is less than about 65% but more than about 6% of totalalpha-1-proteinase inhibitor in the composition.
 36. The composition ofclaim 34, wherein the amount of des-lys alpha-1-proteinase inhibitorthat is less than about 65% but more than about 2% of totalalpha-1-proteinase inhibitor in the composition
 37. A method of treatingfamilial emphysema, the method comprising administering atherapeutically effective amount of a composition of claim
 34. 38. Analpha-1-proteinase inhibitor composition comprising a physiologicallyacceptable carrier and an amount of des-lys alpha-1-proteinase inhibitorthat is more than about 70% of total alpha-1-proteinase inhibitor in thecomposition, the composition made using the method of claim
 1. 39. Amethod of treating familial emphysema, the method comprisingadministering a therapeutically effective amount of a composition ofclaim 38.